In order to facilitate analysis of the complex immune response to cancer cells, a well-defined tumor model system has been established in our group. In this model the metastatic mouse breast cancer 4T1.2, transfected with the viral protein hemagglutinin (4T1.2-HA) as a surrogate tumor antigen, is used. The transfer of the viral HA gene provides a tumor that is much more immunogenic, due to response by CD 8 cytotoxic T cells of the mouse to the viral protein. In addition transgenic mice have been generated where the T cell receptor (TCR) transgene specifically recognizes a single dominant peptide of the viral HA protein (TCR-HA), thus allowing for this antigen-specific T cell population to be accurately followed and monitored in vivo. These mice have been crossed onto strains which lack some of the cytotoxic effector molecules of T cells such as perforin and FasL important for killing tumor cells in vitro. More recently we have used a Nanog-promoter-driven green fluoresence construct to mark 4T1.2-HA breast carcinoma cells with a more cancer stem cell phenotype. Nanog is an embryonic transcription factor that maintains cells in a more primitive pluripotent state. Preliminary data suggest that the 4T1.2-HA-Nanog&gt;GFP+ cells have many characteristics of cancer stem cells both in vitro and in vivo. We now have the ability, using these cells, to analyze how small subpopulations of cancer stem cells may respond to immune-mediated cytotoxic effects as well as standard chemotherapies and molecular targeted therapies, and compare their responses to the bulk cancer cell population. We are extending the Nanog-GFP system in an attempt to isolate cancer stem-cell like populations from various human breast carcinoma cell lines including SUM159 cells, generally regarded as human triple-negative breast cancer cells. In addition we are studying how changes that occur in the tumor microenvironment such as hypoxia, or increased inflammation due to local nitric oxide production, can elicit molecular events in both the cancer cells themselves, as well as immune cells present in the tumor, resulting in tumor progression and the development of metastases.